Seroreactive regions on HPV 16 proteins E1 and E2

ABSTRACT

The invention relates to seroreactive regions on proteins E1 and E2 of human papillomavirus (HPV) 16.  
     The application also relates to a vaccine which contains such peptides which contain the seroreactive regions.  
     The invention likewise embraces compositions for diagnostic purposes which contain peptides with the seroreactive regions.

[0001] The invention relates to seroreactive regions on proteins E1 andE2 of human papillomavirus (HPV) 16.

[0002] The application also relates to a vaccine which contains suchpeptides which contain the seroreactive regions.

[0003] The invention likewise embraces compositions for diagnosticpurposes which contain peptides with the seroreactive regions.

[0004] HPV 16 is one of the human papillomaviruses (Proc. Natl. Acad.Sci., USA 80, 3813-3815 (1983). The organization of the genome of HPV 16has been described in Virology 145, 181-185 (1985).

[0005] Genomic sequences of HPV can be detected in most cases of:preinvasive and invasive cervical tumors. HPV 16 has been identifiedworld-wide as the virus type predominating in these tumors. The HPV 16genome is detectable in more than 50% of cervical tumors, in which caseit is often present integrated into the cellular DNA. Little is knownabout the immune response after infections with HPV 16 or otherpapillomaviruses.

[0006] Initial data: patients suffering from cervical tumors werecompared with healthy individuals with regard to the presence ofantibodies against viral proteins. These viral proteins were then linkedas fusion products with various prokaryotic peptides at their N terminusand then used as antigens in Western blots.

[0007] The object of the present invention is the further identificationof HPV 16 viral structures which can be used as tool in the prophylaxis,diagnosis and therapy of HPV 16-dependent tumorous diseases in humans.The identification of such structures is a prerequisite for thedevelopment of ELISAs which make it possible to test a large quantity ofhuman sera for the presence of HPV 16.

[0008] The present invention therefore embraces seroreactive regions ofthe E1 protein of HPV 16, which have one of the following amino-acidsequences: I. NGWFYVEAVVEKKTGDAISDDENENDSDTGEDLVDFIVNDNDYLT II.NENDSDTGEDLVDFIVND III.MADPAGTNGEEGTGCNGWFYVEAVVEKKTGDAISDDENENDSDTGEDLVDFIVNDNDYLT IV.EDLVDFIVNDNDYLT V. EDLVDFIVNDNDYLTQAETETAHALFTAQEAKQH VI.NENDSDTGEDLVDFIVNDNDYLTQAETETAHALFTAQEAKQHRDAVQVLKRKYL VII. GSPLSDIS;

[0009] seroreactive regions of the E2 protein of HPV 16, which have oneof the following amino-acid sequences: I. DKILTHYENDS II.DKILTHYENDSTDLRDHI III. DLRDHIDYWKH IV. AIYYKAREMGFKHINHQVVPTLA V.AIYYKAREMGFKHINHQVVPTLAVSKNKAL VI. YYKAREMGFKHINHQVVPTLAVSKN VII.INHQVVPTLAVSKNKALQAI VIII. INHQVVPTLAVSKNKAL IX.TLAVSKNKALQAIELQLTLETIYNSQYSNEKWTLQDV X. QLTLETIYNSQYSNEKWTLQDVSLE XI.TLETIYNSQYSNEK XII. TSVFSSNEVSSPEII XIII.VFSSNEVSSPEIIRQHLANHPAATHTKAVALGTEET XIV.EIIRQHLANHPAATHTKAVALGTEETQTTIQRPRSEP XV. TEETQTTIQRPRSEPDTGN.

[0010] The invention furthermore embraces peptides with one or more ofthe seroreactive regions identified above, a vaccine which contains oneor more of the peptides identified above, a composition for diagnosticpurposes for the identification of specific antibodies against HPV E1and/or E2 protein, which likewise contain the peptides identified above,and monoclonal antibodies which have an affinity for one or more of theseroreactive regions of the E1 or E2 protein of HPV 16, and acomposition for diagnostic purposes which contains these monoclonalantibodies.

[0011] In order to identify seroreactive regions in proteins E1 and E2of HPV, the experimental route described in Science 228, 1315-1317(1985) was followed. Subgenomic HPV 16 DNA fragments which had beenrandomly generated by ultrasound treatment and partial DNAse I treatmentwere cloned into the phage vector fuse1 and then expressed as part of aphage coat protein. Seroreactive phage recombinants were identifiedusing sera prepared against E1 and E2, and purified, and theseroreactive regions were characterized by sequencing the HPV 16portion. Polyclonal rabbit sera against an HPV 16 E1 MS2 polymerasefusion protein and against the amino- and carboxyl-terminal part of HPV16 E2 (separate, likewise MS2 fusion proteins) were prepared.

[0012] The filamentous phages embrace the three groups f1, fd and M13.It is common to them all that binding and uptake of the phages takesplace via F pili of the bacteria, i.e. that only F⁺ strains can beinfected. The fd wild-type phage, from which the vector system used isderived, forms particles which are about 900×6 nm in size and which arecomposed in particular of about 2700 subunits of the main coat protein.In addition, in each case 5 molecules of the minor coat proteins pIII,pVI, pvII and pIX are located at both ends of the virions. Thesingle-stranded, circular phage genome which, in the case of the fdwild-type, is 6408 bp in size, carries the information for a total of 10different proteins.

[0013] In the fd derivatives fuse1, fuse2 (Parmley and. Smith, Gene, 7,305-318 (1988)) and fusemm, a tetracycline-resistance gene isintegrated, by insertion of a part of the Tn10 transposon, in the phagegenome, which has been enlarged to about 9.2 kbp in this way. This meansthat the replicative DNA double-stranded phage genomes behave in thebacteria like selectable plasmids and can accordingly be prepared andused for clonings. Another modification from the wild-type is thepresence of a reading frame mutation in the gene for the minor coatprotein pIII in conjunction with an inserted restriction site forcloning expressable DNA fragments. The gene for pIII is composed of twoalmost completely independent domains (Crissmann and Smith, 1984): anN-terminal domain which mediates the binding of the phages to thebacterial cell receptor (F pili) and a C-terminal protein domain whichis responsible for phage morphogenesis. The reading frame mutation,which is located directly behind the signal sequence of the protein,thus leads to inactivation of the gene and accordingly also prevents theformation of infectious particles. This is of importance for thereplication of these phage mutants as plasmids because the fd genomesinactivated in the morphogenesis do not damage the host bacteria (Smith,in: Vectors, A Survey of Molecular Cloning Vectors and Their Uses,Butterworth Publishers, Stoneham, Mass. 61-85, 1987).

[0014] Insertion of suitable DNA fragments and restoration of gene IIIfunctions lead to the formation of infectious phage particles whichcarry additional amino-acid sequences on their coats. These sequencesare accessible to various ligands, for example antibodies, in thenatural state of the phages.

[0015] The fd expression system used in this invention is essentiallybased on setting up phage banks by cloning DNA foreign sequences intothe gene III, and examining the latter with the aid of monoclonal orpolyclonal sera for seroreactive recombinants. An amplification normallytakes place on preparation of these expression banks. The extent of thisreplication of individual clones in turn depends on the nature and sizeof the inserted DNA sequence. This means that different clones differ infrequency, which may differ by up to several powers of ten. It istherefore possible to derive from the stated properties the followingtwo features of the fd expression banks:

[0016] Amplification of the banks, which leads to repeated cloning ofidentical phage clones isolated by immunoscreening.

[0017] Possibility of enriching seroreactive phages by affinitychromatography (columns) because phages in the active state can be boundand eluted again.

[0018] The repeated isolation of identical recombinants was avoided byusing separately set up banks, there being an extremely low probabilityof cloning a DNA fragment prepared identically and in parallel, or ofthe phage-recombinant derived therefrom.

[0019] In this invention, a total of 11 different expression banks forHPV 16 DNA in fuse1 were set up. The number of primary,tetracycline-resistant and insert-harbouring recombinants was in thiscase between 2000 and 90000 per bank. Since complete plasmids composedof about 4 kb vector portion and 8 kb HPV portion in sheared form werealways used for the cloning, the HPV-containing fd recombinants arereduced by about 30%. The fragments cloned in were then expressed, asalready mentioned, as fusion protein of the gene III coat protein. Thecloning site in the gene III is in this case directly behind thetranslated signal sequence for protein export. In order to restore thefunction of the gene it is necessary for an insert to have a definedsize (3n+2; n=0, 1, 2, 3 . . . ). In order to express a defined proteinsequence as fusion protein of the gene III product it is necessary inaddition for both the 3′ and the 5′ junction to be in the correctreading frame, and for the corresponding insert to be present in thecorrect orientation. This therefore applies overall to only about every18th (3×3×2) HPV DNA-containing recombinants. A small portion thereof isin turn inactivated by translation stop codons present in the insert orby proteins which are not functional because of their folding. Becauseof the stated parameters it is difficult to estimate what is the minimumnumber of recombinants necessary to express with great probability anyrequired part of an HPV genome as fd fusion protein in the phage bank.In papillomaviruses about 10 kb of the genome (partly by overlappingopen reading frames) code for proteins. Of 2000 tetracycline-resistantinsert-harboring recombinants, about 100 ({fraction (1/18)}) clonesexpress HPV sequences in a suitable manner. With average HPV fragmentsizes of 50-150 bp, the expressed HPV sequence amounts to about5000-15000 bp. In fact fd banks with about 2000 recombinants prove to besufficiently representative.

[0020] In order to ensure the specificity of the immunoscreening, eitherseveral different recombinants of a seroreactive region or at leastseveral identical but independently isolated phage recombinants werealways isolated.

[0021] The amino-acid position indicators in FIGS. 2 and 5 hereinafterrelate to the E1 and E2 proteins and not to the positions of the openreading frames. The first methionine was given position 1.

EXAMPLE 1

[0022] Preparation of Polyclonal Antisera Against HPV 16 E1

[0023] In order to isolate seroreactive phage recombinants from the HPV16 fd expression bank, initially polyclonal rabbit sera against HPV 16E1 MS2 fusion proteins were prepared. For this, the Pst I A fragment ofHPV 16 (bp 875-3693) was cloned into the Pst I cleavage site of theexpression vector pEX12mer (Seedorf et al., EMBO J. 6, 139-144, 1987),by which amino acids 5-649 of HPV 16 E1 ORF are expressed (FIG. 1). Thisvector is a derivative of the plasmid pPLC24 (Remaut et al., Gene 15,81-93, 1981) which has been-modified by insertion of the pUC8 polylinkerbehind the MS2 polymerase portion. The fusion protein is transcribed inthe pEX12mer by the temperature-inducible lambda pL promoter. TheN-terminal fusion portion of the MS2 protein amounts to 100 amino acids.

[0024] Since the original HPV 16 isolate (Seedorf et al., Virology, 145,181-185, 1985) has a reading frame mutation in the region of the E1 openreading frame (nucleotide position 1138), recourse was had to an HPV 16isolate from a cervical carcinoma with a complete E1 ORF. Because of theselected restriction cleavages, the HPV 16 E1 open reading frame (bp865-2811) is thus completely expressed apart from three N-terminal aminoacids.

[0025] The clonings and plasmid analyses were initially carried outusing the E. coli strain W6 in which there is constitutive expression ofthe repressor for the lambda promoter. This prevented expression of thefusion proteins, in order to prevent counterselection after thetransformations. After examination of the cloning by restrictionanalysis, and Southern blot hybridization with radioactive labelled HPV16 DNA (Pst I A fragment), the plasmid DNA of the construct was used fortransformation in E. coli N6045. This strain is able, because of itstemperature-sensitive repressor of the lambda promoter, to express theMS2 fusion proteins.

[0026] It was then possible in a Western blot to examine, with the aidof a monoclonal antibody directed against the MS2 portion of the fusionprotein, by comparison of extracts from induced and non-induced bacteriathe size and the expression rate of the fusion protein. Since the bandof the MS2 E1 fusion protein corresponded to the expected size of about90 kD, no examination of the cloning junctions by sequencing was carriedout. In the two other reading frames of the HPV 16 Pst I A fragment,expression of larger proteins is impossible because of the presence oftranslation stop codons. In addition, both Pst I cleavage sites of thevector-insert junctions were retained. Correct expression of the E1 openreading frame was confirmed by the results of the immunoscreening of theHPV 16 fd expression banks, which are described in the followingsection.

[0027] The MS2-E1 fusion protein was then purified from induced E. colicultures by differential extraction and by electroelution from SDSpolyacrylamide gels, and was then used to immunize two rabbits.

EXAMPLE 2

[0028] Identification of Seroreactive Regions on the HPV 16 E1 Protein

[0029] Both of the polyclonal rabbit sera prepared against HPV 16 E1were used to examine five different HPV 16 fd expression banks forreactive recombinants. It was possible in this way to identify a totalof at least two different antibody binding sites represented bynon-overlapping phage clones. In total, 19 independent phage cloneswhich contain seven different classes of HPV 16 inserts were isolated(FIG. 2). Six classes have a common overlapping region which codes forthe HPV 16 E1 specific peptide EDLVDFIVND. The second identified epitopeon the E1 protein is represented by a recombinant phage (clone 1059)which codes for the E1 peptide GSPLSDIS.

[0030] The original HPV 16 isolate has a reading frame mutation in theE1 open reading frame (nucleotide position 1138). The DNA of this HPV 16isolate was used to prepare the fd expression banks. Two of the isolatedseroreactive fd recombinants contain this region and therefore also havethe reading frame mutation. In clone 1145 this leads to a change ofreading frame, and this results in C-terminal attachment of three HPV16-E2 non-specific amino acids ( . . . ValValHis). Clone 1059 starts inthe wrong frame and is converted into the correct HPV 16 E1 readingframe by the reading frame mutation of the HPV 16 isolate used. Theclone codes for the peptide STGSRTKVFGSPLKSDIS, of which only theC-terminal amino acids . . . GSPLSDIS derive from the actual HPV 16 E1protein and must form the epitope.

[0031] Both clones which contain the reading frame mutation have thecorrect insert size (3n+2 base pairs) to restore the reading frame ofgene III of the phage vector.

EXAMPLE 3

[0032] Preparation of Polyclonal Antisera Against HPV 16 E2

[0033] Like the case of the HPV 16 E1 open reading frame, no suitableantisera were available for the HPV 16 E2 protein either. For thisreason, the HPV 16 E2 open reading frame (nucleotide position 2756-3850;AA 1-365) was expressed in the vector pEX12mer as already described forthe E1 protein.

[0034] Firstly the HPV 16 DNA fragment was cloned via the Hinf Icleavage site at position 2761 into the pEX12mer vector. In this casethe starting material was an already subcloned HPV 16 fragment (bp2367-4467). This fragment was cut out of the vector again, via theadditionally inserted non-HPV 16-specific restriction sites Xba I (5′end) and Bam HI (3′ end), and prepared. This DNA fragment which is 2.1kb in size (Xba I/Bam HI) was then partially cut with Hinf I. Thisresults, inter alia, in a fragment which is 1700 bp in size between the3′-terminal Bam HI cleavage site and Hinf I site at bp 2761. Theinternal Hinf I cleavage site (bp 3539) in this fragment is uncleaved,and the HPV 16 E2 ORF is completely present apart from threeamino-terminal amino acids. After preparation, the Hinf/Bam fragment wascloned into the pEX12mer expression vector which had been cleaved withBam HI. This resulted, via the compatible Bam HI sites, in linearproducts of vector and insert. The free ends of these products werefilled in with Klenow polymerase and then closed by ligation. Thisresults in an MS2-E2 junction at the filled-in cleavage sites Bam HI(vector) and Hinf I (E2 insert) with loss of the two restriction sites.Using Eco RI/Bam HI double restriction cleavages it was possible toidentify recombinants which harboured the HPV 16 E2 fragment in thecorrect orientation.

[0035] After transformation into the E. coli expression strain 6045 itwas not possible using a monoclonal antibody directed against the MS2polymerase to find any production whatever of the MS2 fusion protein. Inorder to rule out a displacement of the reading frame at the MS2-E2junction, the plasmid DNA of a total of 16 different MS2-E2 recombinantswas hybridized in a Southern blot with an oligonucleotide derived fromthe correct Bam HI/Hinf I junction. Since an unambiguous hybridizationsignal was identifiable with 15 clones, it was assumed that the cloninghad taken place in the correct reading frame, and expression of thecomplete E2 ORP is not possible in pEX vectors. As a substitute, the HPV16 E2 protein was then expressed in two halves in the pEX12mer vector.

EXAMPLE 4

[0036] Expression of the Amino-Terminal Region of HPV 16 E2

[0037] The amino-terminal region of the E2 open reading frame betweennucleotide position 2761 and 3209 was cloned into the pex12mer vectorand expressed. Since the E2 open reading frame starts at nucleotideposition 2756, the MS2-E2 fusion protein lacks the first two amino acids(Met-Glu) of the E2 protein (FIG. 4).

[0038] Plasmid DNA composed of pEX12mer and HPV 16 E2, which wereobtained from the cloning described above, was truncated at the carboxylterminus by deletion of a Hinc II (HPV 16 bp 3209)/Bam HI fragment andreligation (blunt/flush from Hinc II and Bam HI). This results inexpression of the N-terminal part of HPV 16 E2 between nucleotideposition 2761 (Hinf I) and 3209 (Hinc II). A fusion protein about 30 kDin size was detectable in induced bacteria in a Western blot with ananti-MS2 molecule antibody.

[0039] The fusion protein was purified by differential extraction of theinduced bacterial lysate and by electroelution of the protein band fromSDS polyacrylamide gels stained with Coomassie blue, and used forimmunizing rabbits.

EXAMPLE 5

[0040] Expression of the Carboxyl-Terminal Region of HPV 16 E2

[0041] The C-terminal region of the HPV 16 E2 open reading frame betweennucleotide position 3209 and 3850 was expressed in the pEX12mer vector(FIG. 3). The region is thus directly connected to the expressedamino-terminal part, described above, of the HPV16 E2 open readingframe.

[0042] For this, recourse was had to the Xba/Bam fragment which has beendescribed above and which contains the complete HPV 16 E2 reading frame.After restriction cleavage, a Hinc II/Bam HI fragment (nucleotideposition 3209-4467) which contains the carboxyl-terminal half of HPV 16E2 was isolated. This fragment was inserted into the Bam HI cleavagesite of the pEX12mer expression vector (5′BamHI/Hinc II-Bam HI/Bam HI3′). It was possible with the aid of the anti-MS2 monoclonal antibody toidentify in extracts of induced bacteria a fusion protein of about 30kD, which was purified by differential extraction and electroelutionfrom SDS polyacrylamide gels, and was used to immunize rabbits.

EXAMPLE 6

[0043] Identification of Seroreactive Regions on the HPV 16 E2 Protein

[0044] Available for the immunoscreening of the fd HPV 16 expressionbanks was a total of four different anti-HPV 16 E2 antisera: in eachcase two sera against the amino-terminal part (bp 2761-3209; AA 3-152)and two against the carboxyl-terminal part of the E2 (bp 3209-3850; AA153-365) open reading frame. These sera were used to examine fivedifferent expression banks for seroreactive recombinants. This resultedin isolation of a total of 32 clones, of which 26 contain amino-terminalsequences of the E2 protein. These 26 clones form a total of 11different classes which represent four different non-overlapping regions(FIG. 5).

[0045] All the epitopes are located in a restricted region comprising 88amino acids of the amino terminus of the E2 open reading frame which islocated between nucleotide position 2792 (AspLysIle . . . ) and 3055 ( .. . SerLeuGlu).

[0046] It was possible to locate in the carboxyl-terminal region atleast two independent non-overlapping epitopes (TSVFSSNEVSSPEII andTEETQTTIQRPRISEPDTGN, FIG. 5). These are represented by a total of fourclasses of recombinants with six independent isolates. The region of theE2 open reading frame which is covered by the clones is located betweennucleotide position 3343 (ThrSerVal . . . ) and 3502 ( . . . ThrGlyAsn)and comprises 52 amino acids.

[0047] Five classes of recombinants (12 isolates) extend over nucleotideposition 2926. All the clones have a point mutation (A->G transition)here, but this does not lead to a change in the corresponding amino acid(glutamine).

EXAMPLE 7

[0048] Immunoscreening of fd Phage Expression Banks

[0049] 1. Phage Affinity Concentration with Protein A-Sepharose Columns

[0050] The phage banks prepared in the fd phage expression system usedunavoidably underwent amplification on cloning. The extent of thisreplication of the original clones is in turn greatly influenced by thenature of the individual recombinants, for example by different sizes ofinserts or conformation of the coat proteins, inhibition ofphysiological processes in the infected bacteria and many others, and itwas therefore not to be expected that uniform amplification of allphages takes place. In order to isolate underrepresented phagerecombinants or clones from large libraries, seroreactive phagerecombinants were concentrated. For this, use was made of thecircumstance that the foreign sequences expressed in each case appear aspart of an fd gene III fusion protein on the coat of natural phageparticles. Large amounts of phages (10⁹-10¹² particles) were for thispurpose bound to protein A antibody columns and eluted again.

[0051] For this, initially protein A-Sepharose was swollen with PBS for30 min and was washed with PBS. Subsequently the protein A-Sepharose wasincubated with about 1 to 2 ml of suitable polyclonal sera (rabbit orhuman) or with corresponding protein A-binding monoclonal antibodies inEppendorf reaction tubes on a rotary shaker at 4° C. for 1 to 2 days.Subsequently the protein A-Sepharose was washed 10 times by theSepharose being alternately resuspended in 10 ml of PBS and pelletedagain by centrifugation (2 min, 6000 rpm). The protein A-Sepharose-IgGcomplexes formed were then incubated with an appropriate amount ofphages as above. Then the Sepharose was washed with PBS several times asbefore and packed into a Pasteur pipette closed with a glass bead andwashed with several liters. (2-15 l) flowing through. The columnmaterial was removed and then incubated in the same volume of elutionbuffer (1 mg/ml BSA, 0.1 M HCl, glycine, pH 2.2) for 15 min. After briefcentrifugation the supernatant, which now contains free phages andantibodies, was neutralized with ⅕ of the volume of tris base (0.5 M).Antibodies which recognize the recombinant gene are able to inhibitbinding of the phage to the bacterial cells and thus the cycle ofinfection. For this reason the phages were added in 100-200 μl aliquotsof the eluates immediately after neutralization to exponentially growingE. coli K91 and plated out on complete medium plates. It emerged duringthe work that replica filters of these phage platings were unsuitablefor immunoblotting, probably because of contaminants in the eluate. Forthis reason the resulting plaques were again rinsed off the plates withcomplete medium and subsequently plated out from the phage suspensionsobtained in this way, which had undergone renewed amplification, and theimmunoblotting was carried out on minimal agar plates.

[0052] 2. Phage Platings and Preparation of Nitrocellulose ReplicaFilters for the Immunoblotting

[0053] All the fd phage derivatives were plated out on a lawn formed byE. coli K91 (Lyons and Zinder, Virology, 49, 45-60, 1972). This strainis distinguished by a large number of F pili (5 per cell, compared withabout 0.5 per cell in most F⁺ strains) which are responsible for uptakeof filamentous phages. This is particularly important for the fdexpression system used in this study because the recombinant fuse phageshave, owing to the uptake of a part of the Tn10 transposon (tetracyclineresistance), a genome which is distinctly enlarged compared with thewild-type, and for this reason form particularly small plaques.

[0054] To plate out the phages, a K91 overnight culture was diluted1:100 in complete medium (2×YT) and incubated at 37° C. for 3 to 4 h.After a density of E₆₀₀=0.8-1.2 was reached, 200 μl of the bacteria wereplated out with an appropriate amount of phages, together with 3.5 ml ofagarose (0.6% agarose, 10 mM MgSO₄, 50° C.) on prewarmed bacteriaplates. Minimal agar plates were always used for every plating intendedto be used for nitrocellulose replicas for the immunoscreening. Platingsout for determination of the phage titer or for DNA hybridization werecarried out on complete medium plates.

[0055] Use of complete medium plates for the immunoblotting always leadto very high non-specific reactivity of the filters with the sera used.

[0056] The plates were incubated at 37° C. overnight. After about 16hours, a nitrocellulose filter was placed on for 10-15 min, marked withfour asymmetric pricks with a needle and removed again using flat-endedforceps. The filters were labelled and then inverted onto a freshminimal agar plate and incubated further at 37° C. for 5-6 hours. Thisincreased the amount of phage particles (proteins) on the filter sincethe reincubation makes it possible for the bacteria and phages bound tothe filters to grow further via the nutrients diffusing from the plate.Subsequently the filters were removed and saturated in 10% milk (skimmilk powder in PBS) for 30-60 min. The filters were then incubated withsuitable dilutions of appropriate sera in 5% milk at 4° C. overnight.

[0057] 3. Immunostaining of Replica Filters and Cloning of ReactiveRecombinants

[0058] After removal of the replica filters, blocking in 10% milk (inPBS) for 60 min and overnight incubation with antisera, thenitrocellulose filters were washed in PBS, 0.05% Tween 20 (5 changes ofwashing buffer) for 30 min. The filters were then incubated with 1:1000dilutions of appropriate second antibodies (peroxidase-coupled goatanti-human, anti-rabbit or anti-mouse) in 5% milk at RT for 2 h. Thiswas followed by renewed washing (see above) and incubation in thefollowing staining mixture:  40 mg of diaminobenzidine  30 μl of 30%H₂O₂ 1.5 ml of 1% NiSO₄ in 50 ml of PBS

[0059] After sufficient color had developed, the filters were removedfrom the solution, placed in water for 30 min and then dried on 3MMpaper.

[0060] The prick holes and signals on the filters were then copied ontoa sheet or the lid of a bacteria dish. This made it possible to assign aposition or, if the phage dilution was sufficiently large (round 2 orhigher), a plaque to a signal. A sterile toothpick was gently stabbedinto the position or the plaque, and the toothpick was placed in 500 μlof complete medium for 10-15 min. This phage suspension then containedgenerally about 10⁶-10⁷ infectious particles, which comprises about0.1-1% of the phages in a plaque. The phage suspensions were thenincubated at 65° C. for 15-20 min in order to kill bacteria which hadbeen carried over, and were then stored at 4° C.

DESCRIPTION OF THE FIGURES

[0061]FIG. 1

[0062] Cloning of the E1 open reading frame into the expression vectorpEX12mer.

[0063]FIG. 2

[0064] Seroreactive regions on the HPV 16 E1 protein.

[0065] Small letters indicate the amino acids of clones 1145 and 1059which, because of the change in reading frame of the HPV 16 isolate usedfor cloning the fd banks, are not derived from the HPV 16 E1 protein(see text). Clones 1090, 1079, 1084, 1029, 1099 and 1145 have a commonregion of 10 amino acids (EDLVDFIVND) which possibly represents a commonepitope of the clones, although other antibody binding sites on theseclones cannot be ruled out. Clone 1059 has, because of the change inreading frame, no common amino-acid sequences with the other clones,although the insert of this clone overlaps with the insert of clone1145. The position indications relate to the HPV 16 E1 open readingframe. The amino acids of clones 1145 and 1059 which do not derive fromE1 are not taken into account here.

[0066]FIG. 3

[0067] Cloning of the carboxyl-terminal half of the HPV 16 E2 proteininto the expression vector pEX12mer.

[0068]FIG. 4

[0069] Cloning of the amino-terminal half of the HPV 16 E2 protein intothe expression vector pEX12mer.

[0070]FIG. 5

[0071] Seroreactive regions on the HPV 16 E2 protein. The regions(E2-1066, -1170, -1074, -1112) on the carboxyl-terminal half of HPV 16E2 are all located in a region 88 amino-acids long (between AA 13 and100) and partially overlap. The carboxyl-terminal regions are alsoclosely adjacent (between AA 197 and 249). The two regions are in eachcase arranged approximately proportional to their position on the E2protein.

1 44 45 amino acids amino acid linear peptide 1 Asn Gly Trp Phe Tyr ValGlu Ala Val Val Glu Lys Lys Thr Gly Asp 1 5 10 15 Ala Ile Ser Asp AspGlu Asn Glu Asn Asp Ser Asp Thr Gly Glu Asp 20 25 30 Leu Val Asp Phe IleVal Asn Asp Asn Asp Tyr Leu Thr 35 40 45 18 amino acids amino acidlinear peptide 2 Asn Glu Asn Asp Ser Asp Thr Gly Glu Asp Leu Val Asp PheIle Val 1 5 10 15 Asn Asp 60 amino acids amino acid linear peptide 3 MetAla Asp Pro Ala Gly Thr Asn Gly Glu Glu Gly Thr Gly Cys Asn 1 5 10 15Gly Trp Phe Tyr Val Glu Ala Val Val Glu Lys Lys Thr Gly Asp Ala 20 25 30Ile Ser Asp Asp Glu Asn Glu Asn Asp Ser Asp Thr Gly Glu Asp Leu 35 40 45Val Asp Phe Ile Val Asn Asp Asn Asp Tyr Leu Thr 50 55 60 15 amino acidsamino acid linear peptide 4 Glu Asp Leu Val Asp Phe Ile Val Asn Asp AsnAsp Tyr Leu Thr 1 5 10 15 34 amino acids amino acid linear peptide 5 GluAsp Leu Val Asp Phe Ile Val Asn Asp Asn Asp Tyr Leu Thr Gln 1 5 10 15Ala Glu Thr Glu Thr Ala His Ala Leu Phe Thr Ala Gln Glu Ala Lys 20 25 30Gln His 54 amino acids amino acid linear peptide 6 Asn Glu Asn Asp SerAsp Thr Gly Glu Asp Leu Val Asp Phe Ile Val 1 5 10 15 Asn Asp Asn AspTyr Leu Thr Gln Ala Glu Thr Glu Thr Ala His Ala 20 25 30 Leu Phe Thr AlaGln Glu Ala Lys Gln His Arg Asp Ala Val Gln Val 35 40 45 Leu Lys Arg LysTyr Leu 50 8 amino acids amino acid linear peptide 7 Gly Ser Pro Leu SerAsp Ile Ser 1 5 11 amino acids amino acid linear peptide 8 Asp Lys IleLeu Thr His Tyr Glu Asn Asp Ser 1 5 10 18 amino acids amino acid linearpeptide 9 Asp Lys Ile Leu Thr His Tyr Glu Asn Asp Ser Thr Asp Leu ArgAsp 1 5 10 15 His Ile 11 amino acids amino acid linear peptide 10 AspLeu Arg Asp His Ile Asp Tyr Trp Lys His 1 5 10 23 amino acids amino acidlinear peptide 11 Ala Ile Tyr Tyr Lys Ala Arg Glu Met Gly Phe Lys HisIle Asn His 1 5 10 15 Gln Val Val Pro Thr Leu Ala 20 30 amino acidsamino acid linear peptide 12 Ala Ile Tyr Tyr Lys Ala Arg Glu Met Gly PheLys His Ile Asn His 1 5 10 15 Gln Val Val Pro Thr Leu Ala Val Ser LysAsn Lys Ala Leu 20 25 30 25 amino acids amino acid linear peptide 13 TyrTyr Lys Ala Arg Glu Met Gly Phe Lys His Ile Asn His Gln Val 1 5 10 15Val Pro Thr Leu Ala Val Ser Lys Asn 20 25 20 amino acids amino acidlinear peptide 14 Ile Asn His Gln Val Val Pro Thr Leu Ala Val Ser LysAsn Lys Ala 1 5 10 15 Leu Gln Ala Ile 20 17 amino acids amino acidlinear peptide 15 Ile Asn His Gln Val Val Pro Thr Leu Ala Val Ser LysAsn Lys Ala 1 5 10 15 Leu 37 amino acids amino acid linear peptide 16Thr Leu Ala Val Ser Lys Asn Lys Ala Leu Gln Ala Ile Glu Leu Gln 1 5 1015 Leu Thr Leu Glu Thr Ile Tyr Asn Ser Gln Tyr Ser Asn Glu Lys Trp 20 2530 Thr Leu Gln Asp Val 35 25 amino acids amino acid linear peptide 17Gln Leu Thr Leu Glu Thr Ile Tyr Asn Ser Gln Tyr Ser Asn Glu Lys 1 5 1015 Trp Thr Leu Gln Asp Val Ser Leu Glu 20 25 14 amino acids amino acidlinear peptide 18 Thr Leu Glu Thr Ile Tyr Asn Ser Gln Tyr Ser Asn GluLys 1 5 10 15 amino acids amino acid linear peptide 19 Thr Ser Val PheSer Ser Asn Glu Val Ser Ser Pro Glu Ile Ile 1 5 10 15 36 amino acidsamino acid linear peptide 20 Val Phe Ser Ser Asn Glu Val Ser Ser Pro GluIle Ile Arg Gln His 1 5 10 15 Leu Ala Asn His Pro Ala Ala Thr His ThrLys Ala Val Ala Leu Gly 20 25 30 Thr Glu Glu Thr 35 37 amino acids aminoacid linear peptide 21 Glu Ile Ile Arg Gln His Leu Ala Asn His Pro AlaAla Thr His Thr 1 5 10 15 Lys Ala Val Ala Leu Gly Thr Glu Glu Thr GlnThr Thr Ile Gln Arg 20 25 30 Pro Arg Ser Glu Pro 35 19 amino acids aminoacid linear peptide 22 Thr Glu Glu Thr Gln Thr Thr Ile Gln Arg Pro ArgSer Glu Pro Asp 1 5 10 15 Thr Gly Asn 10 amino acids amino acid linearpeptide 23 Glu Asp Leu Val Asp Phe Ile Val Asn Asp 1 5 10 17 amino acidsamino acid linear peptide 24 Ser Thr Gly Ser Lys Thr Lys Val Phe Gly SerPro Leu Ser Asp Ile 1 5 10 15 Ser 51 base pairs nucleic acid doublelinear DNA (genomic) 25 TTGTCATGGG ATCTGAATTC CGGGGGGATC CGTCGACCTGCAGCCAAGCT T 51 17 amino acids amino acid linear peptide 26 Leu Ser TrpAsp Leu Asn Ser Gly Gly Ile Arg Arg Pro Ala Ala Lys 1 5 10 15 Leu 42base pairs nucleic acid double linear DNA (genomic) 27 TTGTCATGGGATCTGAATTC CGGGGGGATC CGTCGACCTG CA 42 14 amino acids amino acid linearpeptide 28 Leu Ser Trp Asp Leu Asn Ser Gly Gly Ile Arg Arg Pro Ala 1 510 33 base pairs nucleic acid double linear DNA (genomic) 29 GGTACCAATGGGGAAGAGGG TACGGGATGT AAT 33 12 amino acids amino acid linear peptide 30Ala Gly Thr Asn Gly Glu Glu Gly Thr Gly Cys Asn 1 5 10 75 base pairsnucleic acid double linear DNA (genomic) 31 TTGTCATGGG ATCTGAATTCCGGGGGGATC CGTCGACCTG CAGGTACCAA TGGGGAAGAG 60 GGTACGGGAT GTAAT 75 25amino acids amino acid linear peptide 32 Leu Ser Trp Asp Leu Asn Ser GlyGly Ile Arg Arg Pro Ala Gly Thr 1 5 10 15 Asn Gly Glu Glu Gly Thr GlyCys Asn 20 25 26 base pairs nucleic acid double linear DNA (genomic) 33TTGTCATGGG ATCTGAATTC CGGGGG 26 10 amino acids amino acid linear peptide34 Leu Ser Trp Asp Leu Asn Ser Gly Gly Ile 1 5 10 30 base pairs nucleicacid double linear DNA (genomic) 35 TTGTCATGGG ATCTGAATTC CGGGGGGATC 3010 amino acids amino acid linear peptide 36 Leu Ser Trp Asp Leu Asn SerGly Gly Ile 1 5 10 27 base pairs nucleic acid double linear DNA(genomic) 37 ACTCTTTGCC AACGTTTAAA TGTGTGT 27 9 amino acids amino acidlinear peptide 38 Thr Leu Cys Gln Arg Leu Asn Val Cys 1 5 57 base pairsnucleic acid double linear DNA (genomic) 39 TTGTCATGGG ATCTGAATTCCGGGGGGATC ACTCTTTGCC AACGTTTAAA TGTGTGT 57 19 amino acids amino acidlinear peptide 40 Leu Ser Trp Asp Leu Asn Ser Gly Gly Ile Thr Leu CysGln Arg Leu 1 5 10 15 Asn Val Cys 30 base pairs nucleic acid doublelinear DNA (genomic) 41 GACTATTATG GTTTATATTA TGTTCATGAA 30 10 aminoacids amino acid linear peptide 42 Asp Tyr Tyr Gly Leu Tyr Tyr Val HisGlu 1 5 10 60 base pairs nucleic acid double linear DNA (genomic) 43TTGTCATGGG ATCTGAATTC CGGGGGGATC GACTATTATG GTTTATATTA TGTTCATGAA 60 20amino acids amino acid linear peptide 44 Leu Ser Trp Asp Leu Asn Ser GlyGly Ile Asp Tyr Tyr Gly Leu Tyr 1 5 10 15 Tyr Val His Glu 20

What is claimed is:
 1. Seroreactive regions on the E1 protein of humanpapillomavirus (HPV) 16, with the following amino-acid sequences: I.NGWFYVEAVVEXKTGDAISDDENENDSDTGEDLVDFIVNDNDYLT II. NENDSDTGEDLVDFIVNDIII. NADPAGTNGEEGTGCNGWFYVEAVVEKKTGDAISDPENENDSDTGE DLVDFIVNDNDYLT IV.EDLVDFIVNDNDYLT V. EDLVDFIVNDNDYLTQAETETAHALFTAQEKQH VI.NENDSDTGEDLVDFIVNDNDYLTQAETETAHALFTAQEAXQHRDA VQVLKRKYL VII. GSPLSDIS.


2. Seroreactive regions on the E2 protein of human papillomavirus (HPV)16, with the following amino-acid sequences: I. DKILTHYENDS II.DKILTHYENDSTDLRDHI III. DLRDHIDYWKH IV. AIYYKAREMGFKHINHQVVPTLA V.AIYYKAREMGFKHINHQVVPTLAVSKNKAL VI. YYKAREMGFKHINHQVVPTLAVSKN VII.INHQVVPTLAVSKNKALQAI VIII. INHQVVPTLAVSKNKAL IX.TLAVSKNKALQAIELQLTLETIYNSQYSNEKWTLQDV X. QLTLETIYNSQYSNEKWTLQDVSLE XI.TLETIYNSQYSNEK XII. TSVFSSNEVSSPEII XIII.VFSSNEVSSPEIIRQHLANHPAATHTKAVALGTEET XIV.EIIRQHLANHPAATHTKAVALGTEETQTTIQRPRSEP


3. A peptide which contains one or more of the seroreactive regions asclaimed in claim
 1. 4. A peptide which contains one or more of theseroreactive regions as claimed in claim
 2. 5. A vaccine which containsone or more of the peptides as claimed in claim
 3. 6. A vaccine whichcontains one or more of the peptides as claimed in claim
 4. 7. Acomposition for diagnostic purposes for identifying specific antibodiesagainst HPV 16 E1 or E2 protein, which contains peptides as claimed inclaim
 3. 8. A composition for diagnostic purposes for identifyingspecific antibodies against HPV 16 E1 or E2 protein, which containspeptides as claimed in claim
 4. 9. A monoclonal antibody which hasaffinity for the seroreactive regions of claim
 1. 10. A monoclonalantibody which has affinity for the seroreactive regions of claim
 2. 11.A composition for diagnostic purposes, which contains a monoclonalantibody as claimed in claim
 9. 12. A composition for diagnosticpurposes, which contains a monoclonal antibody as claimed in claim 10.13. A composition for diagnostic purposes as claimed in claim 11 foridentifying HPV 16 specific E1 or E2 proteins.
 14. A composition fordiagnostic purposes as claimed in claim 12 for identifying HPV 16specific E1 or E2 proteins.
 15. The use of peptides as claimed in claim3 for producing vaccine or for compositions for diagnostic purposes. 16.The use of peptides as claimed in claim 4 for producing vaccine or forcompositions for diagnostic purposes.
 17. An antibody that binds to anamino acid sequence present in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, or SEQ ID NO:22.


18. The antibody of claim 17, wherein the antibody detects or identifiesthe E1 or E2 protein of human papilloma virus 16 (HPV 16).
 19. Theantibody of claim 17, which is a monoclonal antibody.
 20. The antibodyof claim 17, which is a polyclonal antibody.
 21. Polyclonal antiseracomprising the antibody of claim
 17. 22. A composition comprising theantibody of claim
 17. 23. The composition of claim 22, which is adiagnostic composition.
 24. The composition of claim 22, wherein thecomposition is used for identifying, diagnosing, or detecting HPV 16infection.
 25. A method of identifying, diagnosing, or detecting HPV 16infection, said method comprising contacting a sample from a humansuspected of being infected with HPV 16 with an antibody that binds toan amino acid sequence present in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:5, SEQ ID Nb:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, or SEQ ID NO:22, and detecting binding of said antibody to saidamino acid sequence, wherein binding indicates an HPV 16 infection. 26.The method of claim 25, wherein said detecting is by enzyme linkedimmunosorbent assay (ELISA).
 27. The method of claim 25, wherein saidmethod detects binding of said antibody with the E1a, E1b, or E2 proteinof HPV 16.